Food & Function View Article Online
Published on 07 July 2014. Downloaded by University of Chicago on 30/10/2014 21:49:52.
PAPER
Cite this: Food Funct., 2014, 5, 2052
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New insights into the effects of formulation type and compositional mixtures on the antioxidant and cytotoxic activities of dietary supplements basedon hepatoprotective plants a ab b Carla Pereira,a Joa ˜o C. M. Barreira, Ricardo C. Calhelha, Maria Joa ˜o R. P. Queiroz, a a Lillian Barros and Isabel C. F. R. Ferreira*
Artichoke (A), borututu (B) and milk thistle (M) are included in several supplements to provide beneficial effects. Different formulations (infusions, pills and syrups), with different proportions of A, B and M (1 : 1 : 1, 2 : 1 : 1, 1 : 2: 1, 1 : 1 : 2) within each formulation, were assayed to optimize the desired benefits. The antioxidant activity, anti-hepatocellular carcinoma activity, hepatotoxicity and bioactive compound contents were evaluated. Syrups tended to be the formulation with highest antioxidant activity and total phenolic and flavonoid content; otherwise, pills were the worst formulation. In what concerns A : B : M ratios, the results did not reveal so pronounced differences. None of the assayed mixtures resulted to be Received 3rd May 2014 Accepted 6th July 2014
toxic (up to the maximum assayed dose) for liver primary cells (PLP2), but some samples, especially infusions, showed toxicity for the hepatocellular carcinoma cell line (HepG2). With no exception, the
DOI: 10.1039/c4fo00387j
mixtures for all formulations showed synergistic effects on antioxidant activity, when compared to the
www.rsc.org/foodfunction
activity of single plants.
Introduction Artichoke (Cynara scolymus L.), borututu (Cochlospermum angolensis Welw.) and milk thistle (Silybum marianum (L.) Gaertn) are medicinal plants with numerous pharmacological effects, such as antioxidant and hepatoprotective activities, as described in different studies;1–3 borututu is also recognized as an antimalarial herb,4 while milk thistle prevents spleen and gallbladder disorders,5 and artichoke leaves are used for the treatment of dyspepsia and diabetes.6 Phytochemicals are very prone to variations depending on the plant material (that changes within the phenological cycle), harvest, drying and storage conditions.7 The genetic, cultural and environmental factors that explain this variability make their use rather challenging and frequently problematic because the active principles are diverse and may be unknown.7,8 In some cases, these effects might even be harmful; for instance, the leaf extracts of artichoke caused chromosomal instability and cytotoxicity in hamster ovary cells,9 while milk thistle extracts, at 15 mg mL1, showed toxicity against the activity of hepatic P450 cytochrome.10
a Centro de Investigaç˜ ao de Montanha (CIMO), ESA, Instituto Polit´ecnico de Bragança, Campus de Santa Apol´ onia, apartado 1172, 5301-855 Bragança, Portugal. E-mail: [email protected]; Fax: +351-273-325405; Tel: +351-273-303219
Centro de Qu´ımica, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal
b
2052 | Food Funct., 2014, 5, 2052–2060
The consumption of supplements to provide the benecial effects of certain plants has raised several controversial questions, such as those pointed out by Halliwell, who stated that “we cannot just pull out one or two individual molecules and expect pills containing high doses of them to protect us”,11 suggesting the whole herbal medicine as one active ingredient, i.e., a set of multi-component parts self-organized into an indivisible whole.12 Nonetheless, there is an increasing number of formulations based on these plants due to their therapeutic applications, namely infusions, pills, capsules, ampoules, syrups, among others. The bioactivity of the most consumed forms (infusions, pills, and syrups) of borututu, milk thistle, and artichoke was assessed by our research group and these formulations revealed not only antioxidant and hepatoprotective effects, but also synergism between the three plants in syrups.13,14 Thus, given the importance of the studied plants in the treatment of liver diseases and the availability of so many formulations, it seems very pertinent to nd the better way to achieve the desired benets from these herbs depending on the kind of formulation, the plant present in it, or even the percentage of each plant in formulations containing the three mentioned plants. To deepen that question, in the present work we investigated the antioxidant and anti-hepatocellular carcinoma activities of twelve mixtures with four different proportions of artichoke, borututu and milk thistle, and different formulations of each plant (infusions, pills and syrups).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
Experimental
Published on 07 July 2014. Downloaded by University of Chicago on 30/10/2014 21:49:52.
Samples and sample preparation Cynara scolymus L. (artichoke; A), Cochlospermum angolensis Welw. (borututu; B) and Silybum marianum (L.) Gaertn (milk thistle; M) were obtained from a herbalist shop in Bragança (Portugal), as dry materials for infusions (leaves, plant and bark, respectively), pills (based-on plant and roots, in the case of B), and syrups (containing 100% of A, 10% of B roots, and 2.3% of M). Each sample was used as recommended in the label: the infusions were prepared from the dry material and further lyophilized, the pills were powdered and the syrups were directly used. Each formulation (infusions, pills and syrups) of A, B and M, respectively, was mixed in different proportions: 1 : 1 : 1; 2 : 1 : 1; 1 : 2 : 1; and 1 : 1 : 2 (m/m/m), and further dissolved in distilled water to a nal concentration of 6 mg mL1. The twelve stock solutions (four mixtures of infusions, four mixtures of pills and four mixtures of syrups) were successively diluted and submitted to an evaluation of antioxidant activity, anti-hepatocellular carcinoma activity and hepatotoxicity. Standards and reagents 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Gallic acid, catechin, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), ellipticine, phosphate buffered saline (PBS), acetic acid, sulforhodamine B (SRB), trichloroacetic acid (TCA) and Tris were purchased from Sigma (St. Louis, MO, USA). Foetal bovine serum (FBS), L-glutamine, Hank's balanced salt solution (HBSS), trypsin–EDTA (ethylenediaminetetraacetic acid), nonessential amino acid solution (2 mM), penicillin–streptomycin solution (100 U mL1 and 100 mg mL1, respectively) and DMEM (Dulbecco's Modied Eagle Medium) were from Hyclone (Logan, UT, USA). Water was treated in a Milli-Q water purication system (TGI Pure Water Systems, Greenville, SC, USA). Antioxidant activity DPPH radical-scavenging activity was evaluated by using an ELX800 microplate reader (Bio-Tek Instruments, Inc; Winooski, VT, USA), and calculated as a percentage of DPPH discolouration using the formula: [(ADPPH AS)/ADPPH] 100, where AS is the absorbance of the solution containing the sample at 515 nm, and ADPPH is the absorbance of the DPPH solution. Reducing power was evaluated by the capacity to convert Fe3+ into Fe2+, measuring the absorbance at 690 nm in the microplate reader mentioned above. Inhibition of b-carotene bleaching was evaluated though the b-carotene/linoleate assay; the neutralization of linoleate free radicals avoids b-carotene bleaching, which is measured by the formula: b-carotene absorbance aer 2 h of (assay/initial absorbance) 100. Lipid peroxidation inhibition in porcine (Sus scrofa) brain homogenates was evaluated by the decrease in thiobarbituric acid reactive substances (TBARS); the colour intensity of the malondialdehyde-thiobarbituric acid (MDA-TBA) was measured by its absorbance at 532 nm; the inhibition ratio (%) was
This journal is © The Royal Society of Chemistry 2014
Food & Function
calculated using the following formula: [(A B)/A] 100%, where A and B are the absorbance of the control and the sample solution, respectively.15 The results were expressed in EC50 values (sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay). Trolox was used as a positive control. Bioactive compound content Total phenolics were estimated by the Folin-Ciocalteu colorimetric assay according to procedures previously described16 and the results were expressed as mg of gallic acid equivalents (GAE) per g of extract. Total avonoids were determined by a colorimetric assay using aluminum trichloride, following procedures previously reported;16 the results were expressed as mg of (+)-catechin equivalents (CE) per g of extract. Anti-hepatocellular carcinoma activity and hepatotoxicity The anti-hepatocellular carcinoma activity was evaluated using HepG2, which is the most widely used tumor cell line and generally regarded as a good hepatocellular carcinoma model. HepG2 cells were routinely maintained as adherent cell cultures in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U mL1 penicillin and 100 mg mL1 streptomycin, at 37 C, in a humidied air incubator containing 5% CO2. The cell line was plated at 1.0 104 cells per well in 96-well plates. Sulforhodamine B assay was performed according to a procedure previously described by the authors.17 For hepatotoxicity evaluation, a cell culture was prepared from a porcine liver obtained from a local slaughter house, according to a procedure established by the authors;17 it was designed as PLP2. Cultivation of the cells was continued with direct monitoring every two to three days using a phase contrast microscope. Before conuence was reached, cells were subcultured and plated in 96-well plates at a density of 1.0 104 cells per well, and cultivated in DMEM medium with 10% FBS, 100 U mL1 penicillin and 100 mg mL1 streptomycin. The results were expressed in GI50 values (sample concentration that inhibited 50% of the net cell growth). Ellipticine was used as a positive control. Classication of additive, synergistic or antagonistic effects Theoretical values for antioxidant and anti-hepatocellular carcinoma activities of the mixtures were calculated as weighted mean experimental EC50 or GI50 values of the individual samples14 and considering additive contributions of individual species in each percentage; for instance, mixture 2 : 1 : 1: EC50 ¼ EC50A 0.5 + EC50B 0.25 + EC50M 0.25. The classication of additive (AD), synergistic (SN) or antagonistic (negative synergistic; AN) effects was performed as follows: AD: theoretical and experimental values reveal differences lower than 5%; SN: experimental values are more than 5% lower than theoretical values; AN: experimental values are more than 5% higher than theoretical values. For each case, the percentage was calculated as follows: [(experimental value theoretical value)/experimental value] 100. Food Funct., 2014, 5, 2052–2060 | 2053
View Article Online
Food & Function
Paper
In vitro antioxidant properties (EC50, mg mL1) and bioactive compound content for the different formulations prepared from artichoke (A), borututu (B) and milk thistle (M). The results are presented as mean SDa Table 1
Formulation (F)
Published on 07 July 2014. Downloaded by University of Chicago on 30/10/2014 21:49:52.
A : B : M ratio (R)
FR
Infusion Pill Syrup p-value (n ¼ 36) 1:1:1 2:1:1 1:2:1 1:1:2 p-value (n ¼ 27) p-value (n ¼ 108)
Total phenolics (mg GAE g1)
Total avonoids (mg CE g1)
DPPH scavenging activity
Reducing power
b-Carotene bleaching inhibition
TBARS formation inhibition
148 19 69 11 469 164
Published on 07 July 2014. Downloaded by University of Chicago on 30/10/2014 21:49:52.
PAPER
Cite this: Food Funct., 2014, 5, 2052
View Journal | View Issue
New insights into the effects of formulation type and compositional mixtures on the antioxidant and cytotoxic activities of dietary supplements basedon hepatoprotective plants a ab b Carla Pereira,a Joa ˜o C. M. Barreira, Ricardo C. Calhelha, Maria Joa ˜o R. P. Queiroz, a a Lillian Barros and Isabel C. F. R. Ferreira*
Artichoke (A), borututu (B) and milk thistle (M) are included in several supplements to provide beneficial effects. Different formulations (infusions, pills and syrups), with different proportions of A, B and M (1 : 1 : 1, 2 : 1 : 1, 1 : 2: 1, 1 : 1 : 2) within each formulation, were assayed to optimize the desired benefits. The antioxidant activity, anti-hepatocellular carcinoma activity, hepatotoxicity and bioactive compound contents were evaluated. Syrups tended to be the formulation with highest antioxidant activity and total phenolic and flavonoid content; otherwise, pills were the worst formulation. In what concerns A : B : M ratios, the results did not reveal so pronounced differences. None of the assayed mixtures resulted to be Received 3rd May 2014 Accepted 6th July 2014
toxic (up to the maximum assayed dose) for liver primary cells (PLP2), but some samples, especially infusions, showed toxicity for the hepatocellular carcinoma cell line (HepG2). With no exception, the
DOI: 10.1039/c4fo00387j
mixtures for all formulations showed synergistic effects on antioxidant activity, when compared to the
www.rsc.org/foodfunction
activity of single plants.
Introduction Artichoke (Cynara scolymus L.), borututu (Cochlospermum angolensis Welw.) and milk thistle (Silybum marianum (L.) Gaertn) are medicinal plants with numerous pharmacological effects, such as antioxidant and hepatoprotective activities, as described in different studies;1–3 borututu is also recognized as an antimalarial herb,4 while milk thistle prevents spleen and gallbladder disorders,5 and artichoke leaves are used for the treatment of dyspepsia and diabetes.6 Phytochemicals are very prone to variations depending on the plant material (that changes within the phenological cycle), harvest, drying and storage conditions.7 The genetic, cultural and environmental factors that explain this variability make their use rather challenging and frequently problematic because the active principles are diverse and may be unknown.7,8 In some cases, these effects might even be harmful; for instance, the leaf extracts of artichoke caused chromosomal instability and cytotoxicity in hamster ovary cells,9 while milk thistle extracts, at 15 mg mL1, showed toxicity against the activity of hepatic P450 cytochrome.10
a Centro de Investigaç˜ ao de Montanha (CIMO), ESA, Instituto Polit´ecnico de Bragança, Campus de Santa Apol´ onia, apartado 1172, 5301-855 Bragança, Portugal. E-mail: [email protected]; Fax: +351-273-325405; Tel: +351-273-303219
Centro de Qu´ımica, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal
b
2052 | Food Funct., 2014, 5, 2052–2060
The consumption of supplements to provide the benecial effects of certain plants has raised several controversial questions, such as those pointed out by Halliwell, who stated that “we cannot just pull out one or two individual molecules and expect pills containing high doses of them to protect us”,11 suggesting the whole herbal medicine as one active ingredient, i.e., a set of multi-component parts self-organized into an indivisible whole.12 Nonetheless, there is an increasing number of formulations based on these plants due to their therapeutic applications, namely infusions, pills, capsules, ampoules, syrups, among others. The bioactivity of the most consumed forms (infusions, pills, and syrups) of borututu, milk thistle, and artichoke was assessed by our research group and these formulations revealed not only antioxidant and hepatoprotective effects, but also synergism between the three plants in syrups.13,14 Thus, given the importance of the studied plants in the treatment of liver diseases and the availability of so many formulations, it seems very pertinent to nd the better way to achieve the desired benets from these herbs depending on the kind of formulation, the plant present in it, or even the percentage of each plant in formulations containing the three mentioned plants. To deepen that question, in the present work we investigated the antioxidant and anti-hepatocellular carcinoma activities of twelve mixtures with four different proportions of artichoke, borututu and milk thistle, and different formulations of each plant (infusions, pills and syrups).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
Experimental
Published on 07 July 2014. Downloaded by University of Chicago on 30/10/2014 21:49:52.
Samples and sample preparation Cynara scolymus L. (artichoke; A), Cochlospermum angolensis Welw. (borututu; B) and Silybum marianum (L.) Gaertn (milk thistle; M) were obtained from a herbalist shop in Bragança (Portugal), as dry materials for infusions (leaves, plant and bark, respectively), pills (based-on plant and roots, in the case of B), and syrups (containing 100% of A, 10% of B roots, and 2.3% of M). Each sample was used as recommended in the label: the infusions were prepared from the dry material and further lyophilized, the pills were powdered and the syrups were directly used. Each formulation (infusions, pills and syrups) of A, B and M, respectively, was mixed in different proportions: 1 : 1 : 1; 2 : 1 : 1; 1 : 2 : 1; and 1 : 1 : 2 (m/m/m), and further dissolved in distilled water to a nal concentration of 6 mg mL1. The twelve stock solutions (four mixtures of infusions, four mixtures of pills and four mixtures of syrups) were successively diluted and submitted to an evaluation of antioxidant activity, anti-hepatocellular carcinoma activity and hepatotoxicity. Standards and reagents 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). Gallic acid, catechin, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), ellipticine, phosphate buffered saline (PBS), acetic acid, sulforhodamine B (SRB), trichloroacetic acid (TCA) and Tris were purchased from Sigma (St. Louis, MO, USA). Foetal bovine serum (FBS), L-glutamine, Hank's balanced salt solution (HBSS), trypsin–EDTA (ethylenediaminetetraacetic acid), nonessential amino acid solution (2 mM), penicillin–streptomycin solution (100 U mL1 and 100 mg mL1, respectively) and DMEM (Dulbecco's Modied Eagle Medium) were from Hyclone (Logan, UT, USA). Water was treated in a Milli-Q water purication system (TGI Pure Water Systems, Greenville, SC, USA). Antioxidant activity DPPH radical-scavenging activity was evaluated by using an ELX800 microplate reader (Bio-Tek Instruments, Inc; Winooski, VT, USA), and calculated as a percentage of DPPH discolouration using the formula: [(ADPPH AS)/ADPPH] 100, where AS is the absorbance of the solution containing the sample at 515 nm, and ADPPH is the absorbance of the DPPH solution. Reducing power was evaluated by the capacity to convert Fe3+ into Fe2+, measuring the absorbance at 690 nm in the microplate reader mentioned above. Inhibition of b-carotene bleaching was evaluated though the b-carotene/linoleate assay; the neutralization of linoleate free radicals avoids b-carotene bleaching, which is measured by the formula: b-carotene absorbance aer 2 h of (assay/initial absorbance) 100. Lipid peroxidation inhibition in porcine (Sus scrofa) brain homogenates was evaluated by the decrease in thiobarbituric acid reactive substances (TBARS); the colour intensity of the malondialdehyde-thiobarbituric acid (MDA-TBA) was measured by its absorbance at 532 nm; the inhibition ratio (%) was
This journal is © The Royal Society of Chemistry 2014
Food & Function
calculated using the following formula: [(A B)/A] 100%, where A and B are the absorbance of the control and the sample solution, respectively.15 The results were expressed in EC50 values (sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay). Trolox was used as a positive control. Bioactive compound content Total phenolics were estimated by the Folin-Ciocalteu colorimetric assay according to procedures previously described16 and the results were expressed as mg of gallic acid equivalents (GAE) per g of extract. Total avonoids were determined by a colorimetric assay using aluminum trichloride, following procedures previously reported;16 the results were expressed as mg of (+)-catechin equivalents (CE) per g of extract. Anti-hepatocellular carcinoma activity and hepatotoxicity The anti-hepatocellular carcinoma activity was evaluated using HepG2, which is the most widely used tumor cell line and generally regarded as a good hepatocellular carcinoma model. HepG2 cells were routinely maintained as adherent cell cultures in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U mL1 penicillin and 100 mg mL1 streptomycin, at 37 C, in a humidied air incubator containing 5% CO2. The cell line was plated at 1.0 104 cells per well in 96-well plates. Sulforhodamine B assay was performed according to a procedure previously described by the authors.17 For hepatotoxicity evaluation, a cell culture was prepared from a porcine liver obtained from a local slaughter house, according to a procedure established by the authors;17 it was designed as PLP2. Cultivation of the cells was continued with direct monitoring every two to three days using a phase contrast microscope. Before conuence was reached, cells were subcultured and plated in 96-well plates at a density of 1.0 104 cells per well, and cultivated in DMEM medium with 10% FBS, 100 U mL1 penicillin and 100 mg mL1 streptomycin. The results were expressed in GI50 values (sample concentration that inhibited 50% of the net cell growth). Ellipticine was used as a positive control. Classication of additive, synergistic or antagonistic effects Theoretical values for antioxidant and anti-hepatocellular carcinoma activities of the mixtures were calculated as weighted mean experimental EC50 or GI50 values of the individual samples14 and considering additive contributions of individual species in each percentage; for instance, mixture 2 : 1 : 1: EC50 ¼ EC50A 0.5 + EC50B 0.25 + EC50M 0.25. The classication of additive (AD), synergistic (SN) or antagonistic (negative synergistic; AN) effects was performed as follows: AD: theoretical and experimental values reveal differences lower than 5%; SN: experimental values are more than 5% lower than theoretical values; AN: experimental values are more than 5% higher than theoretical values. For each case, the percentage was calculated as follows: [(experimental value theoretical value)/experimental value] 100. Food Funct., 2014, 5, 2052–2060 | 2053
View Article Online
Food & Function
Paper
In vitro antioxidant properties (EC50, mg mL1) and bioactive compound content for the different formulations prepared from artichoke (A), borututu (B) and milk thistle (M). The results are presented as mean SDa Table 1
Formulation (F)
Published on 07 July 2014. Downloaded by University of Chicago on 30/10/2014 21:49:52.
A : B : M ratio (R)
FR
Infusion Pill Syrup p-value (n ¼ 36) 1:1:1 2:1:1 1:2:1 1:1:2 p-value (n ¼ 27) p-value (n ¼ 108)
Total phenolics (mg GAE g1)
Total avonoids (mg CE g1)
DPPH scavenging activity
Reducing power
b-Carotene bleaching inhibition
TBARS formation inhibition
148 19 69 11 469 164
